Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA

Abstract

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and WEISSELLA: Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant's life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.

FIG. 1.

Phylogenetic tree based upon the neighbor-joining method of partial 16S rDNA sequences (E. coli positions 31 to 648) from clones derived by PCR with the primer Bact-0011f and the specific primer Lab-0677r. Suffixes: F, feces; C, cecal chyme; B, baby. Reference sequences are included which were found to be the closest relatives of the retrieved clones. Three subgroups are indicated: I, L. casei-Pediococcus group; II, L. delbrueckii group; III, Leuconostoc paramesenteroides group. B. subtilis is used as an outgroup. The scale bar represents (calculated) distance. The origin of the clones is presented in Table 4.

FIG. 2.

Development of the Lactobacillus-like community over time in an infant. DGGE analysis of amplicons generated by nested PCR from fecal samples taken on day 55 up to day 147. Fragments that are indicated by arrows and numbers were identified by the Lab-clone library of the infant as described in Table 4. The origin of the fragments and the corresponding clone are indicated as follows: 1, L. rhamnosus (B103); 2, L. casei (B121); and 3, L. salivarius (B123).

FIG. 3.

Effect of orally administered L. paracasei F19 on the Lactobacillus-like community over time. DGGE analysis of amplicons generated in a nested PCR with primers Bact-0124-GCf and Uni-0515r. Children were administered L. paracasei F19 (I and II) or placebo (III and IV) for 4 weeks. Fecal samples were taken before the experiment (lanes 0), after 2 weeks of administration (lanes 2), and 2 weeks after administration had ceased (lanes 6). The PCR amplicon for L. paracasei F19 was concomitantly separated. The fragments in the profiles corresponding to F19 are indicated by the box.

FIG. 4.

DGGE analysis of the dominant (d) and Lactobacillus-like community (s) diversity in fecal and cecal samples from six adults (A to D, H, I), after PCR with primers Bact-0124-GCf and Uni-0515r and nested PCR with primers Lab-0159f and Uni-0515-GCr, respectively. The dominant fragments in the Lactobacillus-like patterns indicated by arrows and numbers were identified by clones from the existing clone library of Lactobacillus-like sequences, as described in Table 4. The origin of the fragments and the corresponding clones are indicated as follows: 1, L. sakei (F45); 2, L. ruminis (F68); 3, L. ruminis (F70); 4, L. delbrueckii (F93); 5, L. fermentum (F81); 6, L. ruminis (F1); 7, L. gasseri (S1); and 8, Leuconostoc mesenteroides (S27).

FIG. 5.

Monitoring of the Lactobacillus-like community of adults in time. DGGE analysis of amplicons generated by nested PCR with primers Lab-0159f and Uni-0515-GCr, originating from three individuals (K, L, and M) from whom fecal samples were obtained at 0, 6, and 20 months. The dominant fragments in Lactobacillus-like patterns indicated by arrows and numbers were sequenced and compared to known sequences in GenBank, as described in Table 4. The origin of the fragments and the corresponding clones (see Table 4) are indicated as follows: 1, L. gasseri (F706); 2, L. casei (F703); 3, L. paracasei (F723); 4, L. ruminis (F748); 5, L. ruminis (F754); 6, L. sakei (F749); 7, L. casei (F747); 8, L. ruminis (F761); and 9, L. ruminis (F763).